This application claims benefit of Japanese Application No. 2000-128968 filed on Apr. 25, 2000, the contents of which are incorporated by this reference.
This invention relates to a method of forming a detailed image of cilium and cilia which exists in an epithelial layer of a cellular structure of a bronchi or airway.
As for mucociliary transport, ciliated epithelial cells play an important role as a barrier system for airway structures, such as bronchi or nasal cavities. The ciliated epithelial cells cover the surface of the bronchi. Thus, the abnormalities of ciliated epithelial cells cause various disorders of the trachea bronchial tree.
Ciliated epithelial cells are explained with reference to FIGS. 5 and 6. The cross-sectional view of a layer structure of a bronchial wall is shown in FIG. 5. Moreover, a partial cross-sectional view of a ciliated epithelial cells tissue is shown in FIG. 6.
As shown in FIG. 5, the bronchial wall consists of an epithelial layer 33 which has ciliated columnar epithelial cells 31 and a basement membrane 32, a subepithelial layer 35 which has bundles of elastic fibers 34, a muscle layer 37 which has a smooth muscle (bundle) 36, and an outer muscle layer 39 which is the outer layer of the bronchial wall. The cartilaginous layer 41 consists of cartilage 40, a pericartilaginous layer 42 and bronchial glands 38.
As shown in FIG. 6, the epithelial layer 33 consists of a basement membrane 32, ciliated columnar epithelial cells 31 and cilium 43, with the top of the epithelial layer 33 covered with the gel-like mucus 44. Ciliated columnar epithelial cells 31 are transparent cylinder shaped cells having a nucleus 45 and a length of about 100 xcexcm. The ciliated columnar epithelial cells 32 exist on the basement membrane 32.
Cilium 43 is a colorless and transparent hair-like object with a diameter of 0.1-0.2 xcexcm and length of several xcexcm. Several hundred strands exist at the upper part of each ciliated columnar epithelial cell 31. The gel-like mucus 44 exists from the upper part of the ciliated columnar epithelial cells 31 extended upward by about 6 xcexcm. Furthermore, the upper part of mucus 44 is solid-like. Cilium 43 performs the ciliary motion, a repeated transverse oscillation in the shape of a whip, in the gel-like mucus 44.
Since the epithelial layer 33 is transparent, when the bronchial epithelium of mucus in an organism is observed using an endoscope optical system, an illumination light permeates the cellular structure containing the epithelial layer 33 by about 0.5 mm. For this reason, the bundles of elastic fibers 34 which have color and exist in the cellular structure of a sublayer outside of the epithelial layer 33, are actually observed.
Apart from that, cilium disappears when a cell of bronchial epitheliums becomes cancerous. For this reason, the disappearance of cilium plays an important role in the evaluation of a malignant cell, and location of an affected region.
However, as mentioned above, since ciliated epithelial cells are transparent, it is hard in a conventional method to observe a disappearance of cilium in the process of the cells becoming cancerous, with the naked eye.
The following conventional methods have been used to observe cilia. There is the fluorescent method for observing with an endoscope, wherein an cellular organization extracted from an living body is dyed with a fluorescent paint and a laser light is irradiated onto the cellar organization. There is the method of observing a motion state more indirectly according to a change in the transmittance of a light and the measured frequency of the cilium. There is also the method of observing the scattered light of cilium by the transmitted illumination using a culture cell. In addition, there is the method of observing a change in the input-output signals using the light.
Currently, although it turns out that cilium disappears in the process of becoming cancer, changes in the form and the motion of the cilium caused by other diseases have not been clarified. This is because observation of cilium of a living tissue by the endoscope, etc. was difficult. If a change in the form or motion of cilium of and cilia a living tissue can be clearly discerned and data can be stored, such will become a great assistance in identifying or diagnosing diseases. This is not limited to the specialty of a bronchi or trachea bronchial tree but can also be applied to an otorhinilogy specialty and an obstetrics and gynecology specialty as well.
Moreover, although the observation of a change of the ciliary motion has only been performed in a culture cell, if a cilium observation by the naked eye of an in vivo becomes possible, it could not only confirm the effect of a medicine, but it could also contribute greatly to new medicine development in animal experiments.
Thus, when studying the relationship with a disease, it is important that the ciliary motion in the living body be observed as a direct image.
Conventionally, observing cilium in the living body with the naked eye has not been thought of at all, as mentioned above. In the conventional methods, a microscope, etc. was used to observe a partial cellular structure only after it was collected from an organism and moved to a receptacle so that the cilium of a cellular structure could be observed with the naked eye. However, according to the conventional methods, since the object was observed only about extremely limited parts, the exact range of a wide range observation and a disease could not be specified.
Moreover, since it is indispensable to collect the cellular structure which then becomes an observation object, the physical burden on a subject and the observer""s operation burden becomes significant.
In view of the above mentioned problems inherent in the prior art, it is an object of the present invention to provide a method of forming an image of cilium and its motion in vivo, which can be observed with the naked eye.
Preferred embodiments of the present invention will now be described.
As mentioned above, in an endoscopic observation using any of the conventional observation procedures, the motion of cilium in vivo has not been confirmed with the naked eye.
In an observation screen, a white luminescent point like a halation may be visible accidentally. When it was observed that by raising the observation magnifying power stepwise about this white luminescent point, it has been confirmed that the white luminescent point part was shaking on and off at more than a predetermined magnifying power.
In the conventional observation, an observer had no idea that he was observing a luminescent point at all, since a white luminescent point is recognized as an obstacle to an observation of a subepithelial layer part, and the observation magnifying power in the endoscope was not high enough to confirm the shake of the luminescent point.
However, when this white luminescent point was observed by raising the magnifying power, it was confirmed that this white shake of a luminescent point was none other than the cilium and cilia with motions of the mucous membrane layer due to the motion of the cilia.
The confirmation of the motion of the cilium in vivo by the an endoscope was a very significant achievement scientifically because this was the first time ever that this has been accomplished.
Moreover, an animal and a person""s extracted bronchi were observed by using a stereoscopic microscope. When a stereoscopic microscope was used, it was found that a higher magnifying power (500xc3x97 on a 14 inch monitor) than that of an endoscope was needed in order to observe the cilium. Specifically, the structure (cilium) which by moving causes a halation has been confirmed by further enlarging the part which causes the halation during an animal experiment. In addition, the characteristics of periodicity and polarity were observed in the motion of this structure.
Therefore, cilium can be observed with the naked eye by irradiating illumination light so that a halation may be caused to the surface of ciliated epithelium, guiding the reflected light caused by the halation to an observation optical system, and enlarging the image of the light with a higher observation magnifying power than the predetermined magnifying power.
A method of forming an image of detailed cilium, according to the present invention, wrapped by mucus which exists in surface of epithelial layer of a cellular structure of a bronchus, wherein the method comprises the steps of: irradiating an illumination light to the epithelial layer; generating Fresnel reflected light; irradiating the Fresnel reflected light to an image pick-up optical system; enlarging the Fresnel reflected light by the image pick-up optical system so that the enlarged Fresnel reflected light is made to light-receive into an image pick-up element; scanning at a velocity which is more than the velocity of motion of the cilium by the image pick-up element; transmitting image information from the image pick-up element to an observable medium which can be observed with the naked eye; and displaying the image of detailed cilium and cilia by the observable medium.
Moreover, the step of irradiating an illumination light to the epitheliums layer includes a step of irradiating an illumination light from an upper part to the epithelial layer. In other words, it is not the permeation illumination from a back side but the reflective illumination from the upper part to the epithelial layer. When performing the permeation illumination from a back side with respect to the epithelial layer, it must illuminate from the exterior of an organ if it is performed in the living body, and the body surface must be cut open, and this is not realistic because a considerable burden is imposed on a the patient.
In addition, any type of monochromatic light or white light, may be used for an illumination light.
Moreover, the illumination procedure for creating the halation is as follows. Fresnel reflected light generated at the interface boundary between the cilium and the mucus, due to the difference of the refractive index therebetween, is utilized, and a motion state is observed based on the fluctuation of the reflected light by the ciliary motion.
When performing reflective illumination from the upper part with respect to the epithelial layer, in order to produce the Fresnel reflection broadly, the angle between the incident light of an illumination light and the direct reflected light reflected by the epithelial layer which irradiates to an image pick-up optical system is set to 45xc2x0 or more (preferably 50xc2x0-70xc2x0).
Moreover, in order to obtain the angle between the incident and reflected light, the distance between an observed object and a surface of the image pick-up optical system on an observed object side which is proximate to the observed object is reduced and the inclination angle defined by an illumination optical axis and a reflected light axis is increased, simultaneously with the enlargement of a magnifying power, without changing a parallax of an illuminating system and an observation system. This is suitable when it is required that an outer diameter be small, like an endoscope.
Furthermore, in order to make the outer diameter small, a side-view endoscope can be used as the image pick-up optical system, and the step of irradiating an illumination light from an upper part to the epithelial layer comprises a step of arranging a radiation surface of an illuminating system in a direction where an irradiation angle with respect to the epithelial layer is inclined as much as possible, and the inclination angle between an illumination optical axis and a reflected light axis is increasing (axis of the side-view image pick-up optical system).
Moreover, in case of optical instruments, such as a microscope, with a comparatively large observation distance with respect to an observed object, it is preferable that the axis of an illuminating system is leaned with respect to the axis of an observation system. In this case, if the inclination of the axis of an illuminating system is made variable, it can be adjusted to the angle which tends to cause a Fresnel reflection in accordance with roughness of an observed object. This makes it possible for the inclination angle between the illumination optical axis and the axis of the image pick-up optical system to be variable. If an angle is made variable by the step of measuring an intensity of light which passes through the image pick-up optical system; judging an existence and a level of the Fresnel reflection based on the strength; and adjusting the inclination angle between the illumination optical axis and the axis of the image pick-up optical system, according to the judgment, the Fresnel reflection can be observed to a wide range so that a broad observation can be performed, and the exact range of a disease can be specified.
The step of scanning at a velocity which is more than the velocity of motion of the cilium by the image pick-up element includes a step of providing a high speed camera with the image pick-up element. The high speed camera is defined as that which can obtain an image of 100-10000 sheets per second.
The step of enlarging the Fresnel reflected light by the image pick-up optical system so that the enlarged Fresnel reflected light can be received by the image pick-up element includes a step of providing a high pixel and high resolution system with the image pick-up element. The image pick-up element of a high pixel and high resolution generally has 20-1,000,000 pixels, and more preferably it has more than a 1,000,000 pixels.
The medium which can observe the image by the image pick-up element with the naked eye includes a television monitor. If a television monitor has a common image pick-up element, a very general television monitor is sufficient. If the image pick-up elements are a high speed camera or a high pixel and high resolution system, it is desirable that a scanning speed is high and resolving power is excellent.
The medium observable with the naked eye includes an oscilloscope. On the basis of the electrical signal from the image pick-up element, the frequency of a motion of cilium may be observed not as an image but as a waveform with the naked eye.
When the medium observable with the naked eye includes a television monitor, a display magnifying power of the cilium is 70 magnifications or more.
Further, the method of forming an image of detailed cilium, according to the present invention, comprises a step of providing a recording apparatus for recording the image information simultaneously with the step of transmitting the image information from the image pick-up element to the observable medium with the naked eye. As the recording device, a digital camera, a film camera, a video tape and a video film, a waveform recording device, etc. can be applicable, so that it is convenient to compare a motion of various cilium.